Among the studies recently completed to characterize the role of nitrite as a possible therapeutic option in situations where there is a relative deficiency of nitrite bioactivity are our studies with a mouse model of sickle cell anemia that we have been analyzing for a number of years and our studies in collaboration with the Department of Transfusion Medicine of the NIH Clinical Center on changes in nitrite levels in stored blood. Using mice expressing exclusively sickle hemoglobin, we introduced a novel experimental model of sickle vaso-occlusion. Sickle mice and control mice were challenged with 2 hours of moderate hypoxia with 10% O2 in a normobaric chamber, restored to normoxia, and then 18 hours later had blood sampled for plasma alanine aminotransferase (ALT) as an quantitative measure of tissue injury. Sickle mice showed a 4- fold rise in ALT with this hypoxia-reoxygenation (HR) challenge;wild-type controls or non-sickling colony control mice with the same HR challenge had no change in ALT. Sexual dimorphism was evident such that female mice had 15% higher nitrite levels at baseline, and were less susceptible to HR challenge than males. Therapeutic nitrite supplementation, administered either as 2.4 nanomole/g intraperitoneal injection during HR or oral supplementation for 7 days before HR challenge, abrogated the ALT rise in sickle mice and augmented post-reperfusion complex II-IV mitochondrial respiration.These studies suggest that reduced NO bioavailability in SCD results in dysregulated nitrite homeostasis. Depleted nitrite reserves are associated with enhanced injury with hypoxia-induced vaso-occlusion in this animal model. Therapeutic restoration of nitrite levels, either by intraperitoneal or oral delivery, reduces this injury. In previous studies with human red cells, we found that upon removal of these cells from the body, levels of intracellular nitrite fell rapidly with a half life of less than an hour;we devised a preservation solution using ferricyanide, a thiol reagent and a detergent and could permanetly stabilize these levels. With these methods we found that human red cells normally have a nitrite concentration of about 300 nanomolar, while whole blood levels are about one-half of this, suggesting that most blood nitrite is in erythrocytes. Using these methods we have systematically measured nitrite and nitrate levels in stored whole blood, and red cells both with and without leukoreduction, to see the effects of other components of the blood on nitrite production and or consumption. We find that nitrate levels remain very constant at about 30 micromolar but, to our surprise, we find that the initial rapid fall in nitrite levels tapers and for as long as 42 days significant nitrite levels (about 50 nanomolar) remain in the stored red cells. The levels are comparable in all three methods of storage. We are now conducting studies to establish the mechanism of partial nitrite preservation in stored blood and to see if nitrite supplementation improves the properties of these red cells. In addition, several other studies with long term goals of defining clinical uses of nitrite are being done or being planned at present. We find no evidence of a role of S-nitrosated hemoglobin in the proposed "storage lesion" affecting red blood cells used for transfusion. In collaboration with the NIH Imaging Center we have been examining the effects of changes in nitric oxide levels on blood flow and function in the brains of rodents. We have worked out conditions so that there is no change in systemic or cerebral blood flow with the administration of a nNOS inhibitor but find that their are significant changes in brain function, which is restored with certain nitric oxide donors, including nitrite ions. We are now testing the pharmacological effects of nitrite on brain function and find that nitrite can restore neurovascualar coupling. We have started a project with NHLBI and the DTM of the Clinical Center to study the role of NO depletion in causing painful crises in sickle cell anemia patients. We are measuring levels of hemolysis and evidence of NO destruction by cell-free hemoglobin to see if these parameters correlated with manifestations of the disease. We hve recently received an NIH Bench/Bedside grant for this work and have developed two clinical protocols which are about to be submitted for IRB approval so we can initiate our clinical studies. In our project in collaboration with the American Red Cross to study the role of nitrite ions in the viability and storage of human platelets we have found that there is little change in nitrate levels during five days of room temperature storage and that nitrite levels decrease but only about 50% and then remain stable;some of this appears to be leached from the plastic of the storage bags. We are studying the significance of this finding.